The binding of serum proteins with drugs has long been of interest since it affects the transportation and distribution of pharmaceutical agents in the body. One serum protein that is often involved in this process is α1-acid glycoprotein (AGP). This protein has a carbohydrate content of 45% (w/w). The mean molecular mass for its native form is approximately 41,000 g/mol. AGP has a single polypeptide chain with up to five carbohydrate moieties. It has been estimated that there are 12-20 different forms of AGP in serum due to variations in its amino acid sequence and the types and numbers of carbohydrate groups attached to its polypeptide chain.
Although the exact biological function of AGP is still not clear, the binding of drugs to AGP has long been of interest since this may affect the transport and distribution of such agents in the body. This binding occurs primarily with basic and neutral solutes and is thought to mainly involve hydrophobic interactions; however, coulombic interactions, hydrogen bonding and steric effects may also be present.
Various techniques have been previously used to examine the binding of drugs to AGP. Examples include equilibrium dialysis, ultrafiltration, capillary electrophoresis and various spectroscopic methods. Recent reports with AGP or other serum proteins like human serum albumin (HSA) have also explored the use of high performance affinity chromatography (HPAC) and surface plasmon resonance biosensors for studying the binding of these proteins with drugs. Advantages of these later techniques versus traditional solution-phase methods include their improved precision, their ease of automation, the convenience with which they can provide equilibrium and kinetic information on drug-protein binding, and their ability to reuse the same protein preparation for hundreds of samples.
One desirable feature in the immobilization of a protein for binding studies is to have the protein in a final form that closely mimics the behavior of its native form. It has been demonstrated in many studies based on HPAC that immobilized HSA closely mimics the binding of drugs to the soluble form of this protein. However, previous studies with immobilized AGP have been successful. For instance, reports in which AGP has been immobilized through ionic immobilization and cross-linking have produced materials with a poor correlation versus the behavior of soluble AGP. In addition, no successful reports using currently available commercial AGP columns for drug-protein binding studies have yet been reported, despite the widespread use of such columns for chiral separations.
Therefore, it would be desirable to provide a method of AGP immobilization which produces immobilized AGP which closely mimics the behavior of AGP in its native form.